Modular, multifunctional nanoparticle-based bioconjugate for realtime visualization of cellular membrane potential

ABSTRACT

A construct for detecting cellular membrane potential includes a nanoparticle operable as an electron donor; a modular peptide attached to the nanoparticle, the peptide comprising a nanoparticle association domain, a motif configured to mediate peptide insertion into the plasma membrane, and at least one attachment point for an electron acceptor positioned at a controlled distance from the nanoparticle; and an electron acceptor. The nanoparticle can be a quantum dot and the electron acceptor can be C 60  fullerene. Emission correlates with cellular membrane potential.

CROSS-REFERENCE TO RELATED APPLICATIONS

This Application claims the benefit as a division of U.S. patentapplication Ser. No. 15/882,259 filed on Jan. 29, 2018 which in turnclaims the benefit of U.S. Provisional Patent Application No.62/452,097, the entirety of each of which is incorporated herein byreference.

BACKGROUND

A need exists for techniques to ascertain cellular membrane potential.

BRIEF SUMMARY

A modular, multifunctional nanoparticle (NP)-based electrondonor-acceptor bioconjugate allows for the realtime perception ofchanges in cellular membrane potential. The construct includes thefollowing components: (a) a photoluminescent NP electron donor; (b) amodular, multidomain membrane insertion peptide; and (c) an electronacceptor. The peptide includes (1) a NP association domain, (2) aminoacid motifs to mediate peptide insertion into the plasma membrane, (3)one or more attachment points for attachment of an electron acceptor atdiscreetly controlled locations/distances from the electron donor, and(4) an electron acceptor. The rate of electron transfer between thedonor and acceptor is modulated by changes in membrane potential and theconstruct reports on this modulation by a measurable change in donorphotoluminescence (PL).

A construct made of the above-listed components (a), (b), and (c) wasdelivered to the plasma membrane of living cells. The membrane potentialwas changed by addition of potassium chloride (KCl) and the opticalchanges in donor PL were recorded. The efficiency of donor PL modulation(quenching) by changes in membrane potential was shown to track with thedonor-acceptor separation distance (controlled by peptide design) andthe degree of KCl-induced membrane depolarization.

In a first embodiment, a construct for detecting potentials comprises ananoparticle operable as an electron donor; a modular peptide attachedto the nanoparticle, the peptide comprising a nanoparticle associationdomain, a motif configured to mediate peptide insertion into the plasmamembrane, and at least one attachment point for an electron acceptorpositioned at a controlled distance from the nanoparticle; and anelectron acceptor.

In another embodiment, a method of detecting membrane potentialcomprises providing a construct according to the first embodiment;contacting a cell with the construct; and detecting emission from theconstruct, wherein the emission correlates with cellular membranepotential.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C show nanoparticle (NP)-based membrane-insertingdonor-acceptor electron construct for sensing membrane potential. FIG.1A is a general scheme of a NP-peptide-electron acceptor assembly. Theconstruct includes (1) a core NP electron donor scaffold with a (2)hydrophilic coating. A modular peptide (3) is self-assembled to the NPsurface via a NP-binding domain (4). The peptide further contains amembrane-insertion domain (5) for the insertion of the peptide into theplasma membrane bilayer and has multiple points of attachment for anelectron acceptor (6). FIG. 1B is an example schematic in which the NPis a quantum dot (QD), the peptide is a helical-forming peptide that hasmultiple points for the covalent attachment of a C₆₀ fullerene electronacceptor thus allowing control over the donor-acceptor distance and, asa result, the rate of electron transfer from the donor to acceptor. AHis₆ domain drives assembly of the peptide to the QD surface. FIG. 1Cillustrates that at resting potential, the electrons from thephoto-excited QD donor are attracted to the positively-charged outerleaflet of the membrane which keeps the QD photoluminescence (PL) on(“QD on”). When the membrane potential reverses (depolarized), electronsfrom the photoexcited QD are attracted to the positively-charged innerleaflet of membrane and the C₆₀ electron acceptor, which quenches the QDPL (“QD off”).

FIGS. 2A and 2B illustrate models of QD-peptide-C₆₀ conjugates showsdistance dependent positioning of C₆₀ and the role of ligand in C₆₀accessibility. Shown is 605 nm-emitting QD capped with (A) DHLA or (B)DHLA-PEG750-OMe ligands. In each case the peptides JBD-1, JBD-2 andJBD-3 are assembled to the QD surface and show the distance-dependentnature of the position of the C₆₀ acceptor afforded by the attachment ata unique lysine residue within each peptide sequence. QDs coated withDHLA ligands permit solvent accessibility of the C₆₀ moiety whendisplayed by each of the three peptides while only JBD-3 allows solventdisplay of the C₆₀ on DHLA-PEG750-OMe-capped QDs. Peptide JBD-1 appendsthe C₆₀ closest to the QD center while peptide JBD-3 positions the C₆₀at the most distal position.

FIGS. 3A-3D show steady state quenching of QD PL by peptide-C₆₀ isdependent on peptide ratio and QD-C₆₀ separation distance. Each peptidewas assembled onto a fixed amount of QD at the ratios shown and spectracollected. Shown are the quenching spectra for C₆₀ peptide conjugates of(A) JBD-1, (B) JBD-2 and (C) JBD-3 on DHLA-capped QDs. The quenchingefficiency for each peptide as a function of peptide ratio (valence) isshown in FIG. 3D. The data show the valence- and C₆₀ distance-dependentnature of the efficiency of peptide-C₆₀-mediated QD quenching.

FIG. 4 shows confocal laser scanning microscopy (CLSM) images of A549cells stained with QD-peptide-C₆₀ complexes. Shown are differentialinterference contrast (DIC) (left) and confocal microscopy images(right) showing fluorescence emission of QD-peptide-C₆₀ bound to A549cells. The cells were incubated with 20 nM QD complexed with JBD-1-C60peptides (QD:peptide ratio 1:20) in PBS (pH 8.2) for 10 min at 37° C.For imaging the samples were excited at QD excitation at 402 nm withfluorescence detection channel set to 570-620 nm (red). A dichroicmirror at 561 nm was used to reflect incident excitation light.

FIGS. 5A and 5B are confocal images and time-resolved quantification offluorescence emission of live HeLa cells in resting and depolarizingstate using different potassium chloride (KCl) solutions, respectively.FIG. 5A shows DIC (left) and confocal images (right) of cells labeledwith QD-JBD1-C₆₀ and imaged in an isotonic solution containing 2.5 mMKCl or 140 mM KCl. The samples were excited at 402 nm with afluorescence detection channels set to 570-620 nm. (red) with dichroicmirrors at 561 nm. FIG. 5B shows time-resolved quantification offluorescence emission live HeLa cells labeled with commercial FluoVolt,QD-peptide-C₆₀ and or QD alone. Image collection began at t=0 when cellswere in 100% isotonic solution containing 2.5 mM KCl (0.15 mL). Isotonicsolution containing 140 mM KCl was perfused onto the cells at a rate of˜1.0 mL/min (˜3 mL total volume added). Fluorescence images werecollected every 20 s for 5 min. Fluorescence quantification was done bysampling multiple regions of interest (ROI) in individual cells for eachpeptide construct. The data represent multiple ROIs from 50-70 cellsfrom 4 independent experiments for each QD-peptide sample.

DETAILED DESCRIPTION

Definitions

Before describing the present invention in detail, it is to beunderstood that the terminology used in the specification is for thepurpose of describing particular embodiments, and is not necessarilyintended to be limiting. Although many methods, structures and materialssimilar, modified, or equivalent to those described herein can be usedin the practice of the present invention without undue experimentation,the preferred methods, structures and materials are described herein. Indescribing and claiming the present invention, the following terminologywill be used in accordance with the definitions set out below.

As used herein, the singular forms “a”, “an,” and “the” do not precludeplural referents, unless the content clearly dictates otherwise.

As used herein, the term “and/or” includes any and all combinations ofone or more of the associated listed items.

As used herein, the term “about” when used in conjunction with a statednumerical value or range denotes somewhat more or somewhat less than thestated value or range, to within a range of ±10% of that stated.

Overview

The controlled interfacing of nanoparticle (NP) materials with cellularsystems has been employed for a variety of applications includingcellular labeling/tracking, imaging, sensing, and drug delivery.NP-biological hybrid bioconjugates (e.g., semiconductor quantum dots(QDs) appended with functional peptides or proteins) are value-addedmaterials wherein the ensemble bioconjugate performs a function notattainable by the individual component materials alone. Such constructsbased on energy or charge transfer have much to offer in the developmentof optical or opto-electrical sensors for assessing cellular states(e.g., health, disease, membrane potential). Sensors aimed at reportingon the electrical activity/membrane potential of cells are critical forunderstanding the communication process amongst neurons in the brain(brain mapping) as well as for the assessment of the activity of otherelectrically active cell types (e.g., muscle cells). To date, mostopto-electrical sensors for imaging cellular membrane potential arebased on either (1) stand-alone electrochromic voltage-sensitive dyes(VSDs) or (2) molecular wires based on electron transfer quenching. VSDsare plagued by poor solubility in aqueous media, nonspecific labeling ofnon-membrane cellular structures, poor photostability and inherentcytotoxicity. Molecular wires require intricate molecular synthesis andpurification strategies, the incorporation of a tailored molecular “wirebridge” to conduct electrons from donor to acceptor, and often usepoorly photostable molecular fluorophores (e.g., fluorescein) as theoptical readout moiety. Cumulatively, these issues represent significantlimitations of currently available electro-optical materials foroptically sensing membrane potential.

Described herein is a bioconjugate including a modular, multifunctionalnanoparticle-based electron donor-acceptor bioconjugate for the realtimeperception of changes in cellular membrane potential. The constructcomprises the following components: (a) a photoluminescent NP electrondonor, (b) a modular, multidomain membrane insertion peptide, and (c) anelectron acceptor (FIG. 1A). The peptide includes (1) a NP associationdomain, (2) amino acid motifs to mediate peptide insertion into theplasma membrane, (3) one or more attachment points for attachment of anelectron acceptor at discreetly controlled locations/distances from theelectron donor, and (4) an electron acceptor. The rate of electrontransfer between the donor and acceptor is modulated by changes inmembrane potential and the construct reports on this modulation by ameasurable change in donor photoluminescence (PL). FIG. 1B shows aspecific example of the donor-acceptor bioconjugate where the electrondonor is a semiconductor quantum dot (QD) that is connected to anelectron acceptor, a carbon allotrope (C₆₀ or fullerene) via amembrane-insertion peptide. FIG. 1C shows a schematic representation ofthe QD-peptide-C₆₀ construct reporting on the transition of the cellfrom resting potential state (QD signal “on”) to depolarized state (QDsignal “off”)

EXAMPLES

Peptides of varying lengths and attachment points for the C₆₀ fullereneelectron acceptor were synthesized and iteratively tested for theirability to quench the excited state QD donor (Table 1). These peptideseach contain a His₆ tract to mediate attachment of the peptide to theZnS shell of the QD. Each peptide also bears a unique lysine residue forthe covalent attachment of the C₆₀ to position the C₆₀ moiety atdiscreetly controlled distances from the QD donor center. The peptidesin Table 1 have sequence identification as follows: JBD-1 is SEQ ID No:1; JBD-2 is SEQ ID No: 2; and JBD-3 is SEQ ID No: 3.

TABLE 1 Multifunctional modular peptides for membrane insertion and QD-coupled membrane potentialsensing Peptide Sequence* JBD-1Ac-AAAALAAAAALAALAKAAAAGGH₆-COOH JBD-2 Ac-KAAALAAAAALAAWAALAAAGGH₆-COOHJBD-3 Ac-KAAALAAAAALAAWAALAAAP₉GGH₆-COOH *N terminus blocked with anacetyl group; C terminus blocked with an amide K = unique lysine forattachment of C₆₀ electron acceptor

Peptides of varying lengths and attachment points for the C₆₀ fullereneelectron acceptor were synthesized and iteratively tested for theirability to quench the excited state QD donor (Table 1). These peptideseach contain a His₆ tract to mediate attachment of the peptide to theZnS shell of the QD. Each peptide also bears a unique lysine residue forthe covalent attachment of the C₆₀ to position the C₆₀ moiety atdiscreetly controlled distances from the QD donor center.

Molecular modeling of the orientation of the three peptide species onceassembled to the QD via the His₆ tract revealed the predictedorientation and distance of the C₆₀ acceptor from the QD center whenpresented to the QD as a covalently attached moiety to the peptidebackbone. In the progression from peptide JBD-1 to JBD-3, the C₆₀acceptor is positioned at increasingly further distances from the QDcenter (FIG. 2). It is clear from the modeling that the nature of thehydrophilic ligand on the QD surface impacts the solvent accessibilityof the C₆₀ moiety. QDs coated with dihydrolipoic acid (DHLA) ligandsshowed complete display of the C60 by peptides JBD-2 and JBD-3 andpartial display in the context of peptide JBD-1. QDs coated withDHLA-PEG750-OMe (pegylated DHLA capped with a terminal methoxy group)showed display of the C₆₀ only in the context of peptide JBD-3 while inthe other two peptide species the C₆₀ was buried within the ligandlayer. As the DHLA-coated QDs mediated better cell binding thanDHLA-PEG750-OMe, DHLA QD-peptide-C₆₀ complexes were used for cellularassays.

Steady state fluorescence measurements were performed to determine theefficiency of electron transfer between the QD and the variouspeptide-C₆₀ species. The readout for the assay is the quenching of QD PLupon attachment of the peptide-C₆₀ conjugate to the QD surface. Two keyparameters were assessed for their role in affecting efficient quenchingof the photo-excited QD donor: (1) peptide-C₆₀ valence (or number ofpeptide-C₆₀ conjugates arrayed around the QD surface) and (2) thedistance of the C₆₀ electron acceptor from the QD donor center. As shownin FIG. 3 each peptide-C₆₀ species displayed the ability to efficientlyquench the PL emission of the QD donor in a ratiometric manner (i.e.,the quenching efficiency increased as the number of peptides arrayedaround the QD increased).

Next, fluorescence imaging was used to confirm the successful labelingof the plasma membrane with the QD-peptide-C₆₀ conjugates. A549 (humanlung adenocarcinoma) cells were labeled with 605 QD-DHLA-peptide-C₆₀conjugates and compared to cells incubated with QD alone. FIG. 4 showsDIC and corresponding fluorescence micrographs of A549 cells labeledwith the various QD-peptide-C₆₀ constructs. The cellular labeling isclearly membranous, consistent with the insertion of the peptide-C₆₀moiety into the plasma membrane and the docking of the QD onto thesurface of the plasma membrane. Immuno-silver staining assays confirmedthe insertion of the C₆₀ moiety into the aliphatic portion of the plasmamembrane.

The ability of the QD-peptide-C₆₀ systems to visualize changes inmembrane potential was confirmed by performing depolarizationexperiments on HeLa (human cervical carcinoma) cells that were labeledwith the QD-peptide-C₆₀ conjugates. The cells were depolarized with anisotonic solution containing 140 mM potassium chloride (KCl). Incubationof cells in this solution causes depolarization of the cells by theinflux of K⁺ ions through K⁺ leak channels. FIG. 5A shows representativeconfocal fluorescence images for cells labeled with QD-JBD-1-C₆₀conjugates before and after the addition of the KCl solution.

The images clearly show the time-resolved reduction in QD PL uponperfusion of the KCl solution onto the cells and subsequentdepolarization. Similar responses for QD-peptide conjugates of JBD-2-C₆₀and JBD-3-C₆₀ were obtained (images not shown). A graph of the resultingtime-resolved PL intensities in response to depolarization is shown inFIG. 5B. Minimal change in PL of QDs not decorated with the peptide-C₆₀conjugates was noted, clearly demonstrating the dependence of theresponse on the presence of the peptide-C₆₀ conjugate. Also included isa comparison of QD-peptide-C₆₀-derived cellular response to thatobtained in HeLa cells labeled with a “state-of-the-art” molecularwire-based membrane potential probe (FluoVolt; commercially availablefrom ThermoFisher). For comparison, the QD-JBD-1-C₆₀ conjugate exhibiteda maximum response (PL decrease) of 31% at 5 min while the FluoVoltprobe displayed a fluorescence response of 19% (PL increase) over thissame time course. These data clearly demonstrate the superiorsensitivity of the QD-JBD-1-C₆₀ conjugate compared to the commercialprobe. Further, the magnitude of the QD PL response exhibited a distinctdistance dependent response to depolarization. The PL response trackedinversely with the separation distance between the QD donor center andthe C₆₀ moiety, in good agreement with the plate-based steady state datapresented in FIG. 3.

Concluding Remarks

Potential application areas include those where stable, long termimaging of changes in membrane potential are desired, for exampleimaging/optical recording of the electrical activity in one or morecultured cells, or in tissue slices, whole issues, and/or animals.Targeted cell types in these applications would include (but are notlimited to) electrically active cells such as neurons and muscles cells.These material constructs could also find utility in quantum dot-basedLED cells where the tuning of QD luminescence in the presence of anelectric field is desired/required.

Advantages of NP-peptide-acceptor assemblies as described herein includethe following. They are potentially amenable to both covalent andnoncovalent attachment strategies. Modular design of functional domainsallows for flexibility in iterative peptide development and testing. Thestrategy is amenable to the assembly of conceivably any class of NP withany modular, multifunctional polymer. The NP surface can befunctionalized with different types of modular, multifunctional peptides(“mixed” surfaces) giving ratiometric control over the nature of thedecorated NP surface and iterative control over the rate ofdonor-acceptor electron transfer

More particular advantages of the QD-peptide-C₆₀ construct describedherein include the following. The peptide self-assembles noncovalentlyto the QD donor surface without the need for complex covalent attachmentchemistries that use high concentrations of excess reactants thatrequire purification. The peptide assembles to the QD rapidly (10 min)with high (nM) affinity. The assembled construct labels cell membranesrapidly (10 minutes) after conjugate assembly, with 20 minutes totaltime for cellular labeling with the QD-peptide constructs. The peptidelinker does not conduct, shuttle or otherwise direct the transfer ofelectrons from the donor to the acceptor which simplifies the design andsynthesis of QD-C60 linkers. Electron transfer is completelydistance-dependent which is a key distinguishing factor relative toother membrane potential imaging molecular wires (e.g., FluoVolt sold byThermoFisher), and this dependency can be iteratively controlled bycontrolling the donor-acceptor separation distance. Furthermore, theratio or valence (and thus the avidity) of the peptide for the NP can becontrolled and can be used to tune the rate or efficiency of donorquenching/electron transfer. The exceptional photo stability of QD-basedconstructs allows for much longer imaging times (>100×) compared tovoltage-sensitive dyes. The significantly large two-photon action crosssection of QD materials (10²-10³ greater than organic dyes) makes themideal for deep tissue imaging.

Although the present invention has been described in connection withpreferred embodiments thereof, it will be appreciated by those skilledin the art that additions, deletions, modifications, and substitutionsnot specifically described may be made without departing from the spiritand scope of the invention. Terminology used herein should not beconstrued as being “means-plus-function” language unless the term“means” is expressly used in association therewith.

All documents mentioned herein are hereby incorporated by reference forthe purpose of disclosing and describing the particular materials andmethodologies for which the document was cited.

REFERENCES

Assembly of Peptides Onto the Surface of QDs and QD-Based ChargeTransfer

-   Stewart, M. H., et al., Competition between Förster resonance energy    transfer and electron transfer in stoichiometrically assembled    semiconductor quantum dot-fullerene conjugates. ACS Nano, 2013.    7(10): 9489-9505.-   Medintz, I. L. et al., Quantum-dot/dopamine bioconjugates function    as redox coupled assemblies for in vitro and intracellular pH    sensing. Nat. Mater. 2010. 9: 676-684.-   Mao, C., et al., Viral assembly of oriented quantum dot nanowires.    Proc Natl Acad Sci USA, 2003. 100(12): p. 6946-51.-   Medintz, I. L., et al., A reactive peptidic linker for    self-assembling hybrid quantum dot-DNA bioconjugates. Nano    Lett, 2007. 7(6): p. 1741-8.-   Medintz, I. L., et al., Intracellular Delivery of Quantum    Dot-Protein Cargos Mediated by Cell Penetrating Peptides. Bioconjug    Chem, 2008.-   Slocik, J. M., et al., Peptide-assembled optically responsive    nanoparticle complexes. Nano Lett, 2007. 7(4): p. 1054-8.-   Soman, C. P. and T. D. Giorgio, Quantum dot self-assembly for    protein detection with sub-picomolar sensitivity. Langmuir, 2008.    24(8): p. 4399-404.-   Susumu, K, et al., Enhancing the stability and biological    functionalities of quantum dots via compact multifunctional ligands.    J Am Chem Soc. 2007 129(45):p 13987-96.-   Sapsford K, et al., Kinetics of metal-affinity driven self-assembly    between proteins or peptides and CdSe—ZnS quantum dots. J. Physical    Chem. C. 2007. 111:p. 11528-11538.    Cellular Uptake of and Membrane Labeling with QD-Peptide Assemblies-   Delehanty, J. B. et al. Delivering quantum-dot peptide bioconjugates    to the cellular cytosol: escaping from the endolysosomal system.    Integrat. Biol. 2010. 2:265-277.-   Boeneman, K., et al., Selecting improved peptidyl motifs for    cytosolic delivery of disparate protein and nanoparticle materials.    ACS Nano. 2013. 7(5): 3778-3796.-   Rozenzhak, S. M., et al., Cellular internalization and targeting of    semiconductor quantum dots. Chemical Communications, 2005(17): p.    2217-2219.-   Derfus, A. M., W. C. W. Chan, and S. N. Bhatia, Intracellular    delivery of quantum dots for live cell labeling and organelle    tracking. Advanced Materials, 2004. 16(12): p. 961-+.-   Lagerholm, B. C., et al., Multicolor coding of cells with cationic    peptide coated quantum dots. Nano Letters, 2004. 4(10): p.    2019-2022.-   Ruan, G., et al., Imaging and tracking of tat peptide-conjugated    quantum dots in living cells: new insights into nanoparticle uptake,    intracellular transport, and vesicle shedding. J Am Chem Soc, 2007.    129(47): p. 14759-66.-   Lei, Y., et al., Applications of mesenchymal stem cells labeled with    Tat peptide conjugated quantum dots to cell tracking in mouse body.    Bioconjug Chem, 2008. 19(2): p. 421-7.-   Chang, J. C., H. L. Su, and S. H. Hsu, The use of peptide-delivery    to protect human adipose-derived adult stem cells from damage caused    by the internalization of quantum dots. Biomaterials, 2008.    29(7): p. 925-36.-   Lieleg, O., et al., Specific integrin labeling in living cells using    functionalized nanocrystals. Small, 2007. 3(9): p. 1560-5.-   Shah, B. S., et al., Labeling of mesenchymal stem cells by    bioconjugated quantum dots. Nano Lett, 2007. 7(10): p. 3071-9.-   Biju, V., et al., Quantum dot-insect neuropeptide conjugates for    fluorescence imaging, transfection, and nucleus targeting of living    cells. Langmuir, 2007. 23(20): p. 10254-61.-   Lieleg, O., et al., Specific integrin Labeling in living Celts using    functionalized nanocrystals. Small, 2007. 3(9): p. 1560-1565.-   Medintz, I. L., et al., Intracellular Delivery of Quantum    Dot-Protein Cargos Mediated by Cell Penetrating Peptides. Bioconjug    Chem, 2008. 19: 1785:1795.    Membrane-Inserting Peptides-   Boeneman, K., et al., Selecting improved peptidyl motifs for    cytosolic delivery of disparate protein and nanoparticle materials.    ACS Nano. 2013. 7(5): 3778-3796.    Optical Molecular Wires for Visualizing Changes in Membrane    Potential-   Woodford, C. R., et al. Improved PeT molecules for optically sensing    voltage in neurons. J. Am. Chem. Soc.-   2015. 137: 1817-1824.-   Huang, Y-L., et al. A photostable silicon rhodamine platform for    optical voltage sensing. J. Am. Chem. Soc.-   2015. 137: 10767-10776.-   Davis et. al. Molecular-wire behavior in p-phenylenevinylene    oligomers. Nature 1998. 396: 60-63.

What is claimed is:
 1. A construct for detecting cellular membranepotential, comprising: a nanoparticle operable as an electron donor; amodular peptide attached to the nanoparticle, the peptide comprising ananoparticle association domain, a motif configured to mediate peptideinsertion into a plasma membrane, and at least one attachment point foran electron acceptor positioned at a controlled distance from thenanoparticle; and an electron acceptor, wherein the modular peptide isselected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, andSEQ ID NO:
 3. 2. The construct of claim 1, wherein the nanoparticle is aquantum dot having a hydrophilic coating.
 3. The construct of claim 1,wherein the electron acceptor is C₆₀.